MicroRNAs play crucial roles regulating diverse biological processes in animals, but the mechanisms by which microRNAs themselves are regulated are largely unknown. Recent studies from the Ambros laboratory and others suggest that microRNAs are regulated at many levels post-transcriptionally, including primary to mature microRNA processing, RISC activity, and microRNA turnover. The research proposed here aims to elucidate post-transcriptional regulation of microRNAs using C. elegans as a model system, with a particular focus on microRNA turnover. First, I will develop transgenic worm strains that express two fluorescent reporters to directly monitor changes in post- transcriptional regulation of let-7 or mir-35-41 by simultaneously tracking microRNA target repression and transcriptional activity of the microRNA promoter. I will use these strains to conduct classical genetic screens as well as candidate-driven RNAi screens to identify genes that modify microRNA target repression but not primary microRNA transcription. Second, I will characterize the roles of candidate genes in microRNA turnover, with an emphasis on studying XRN-2. I will perform RNAi against XRN-2 and other candidates, in order to determine their direct impact microRNA stability, define which microRNAs they regulate, and determine whether they thus play a biological role in any previously-characterized process involving microRNAs in vivo. The studies proposed here will begin to elucidate the mechanisms that regulate microRNA stability, and will likely open up multiple new directions of study in the field. Overall, our basic comprehension of these processes will be crucial for the understanding of human disease states, when integrated with other levels of gene expression control. PUBLIC HEALTH RELEVANCE: The expression of microRNAs is deregulated in many human disease states, including cancer. Though the transcription and biogenesis of microRNAs has been studied in depth, very little is known about microRNA turnover, which could be equally important in regulating microRNA levels. Therefore, an understanding of post-transcriptional regulation of microRNAs, and microRNA turnover specifically, will be crucial to advancing our comprehension of gene expression changes that underlie human disease.